Plasmodium falciparum antigens inducing protective antibodies

ABSTRACT

A molecule, protein or peptide characterized in that it is recognized by cytophilic antibodies from individuals who are immune to infection by Plasmodia, and recognized by non-cytophilic antibodies from individuals who are vulnerable to infection by Plasmodiae. Said antibodies are capable of blocking the erythrocytic phase of the parasite by co-operating with accessory cells such as monocytes.

This application is a Division of application Ser. No. 08/416,711, filedOct. 8, 1993, now U.S. Pat. No. 6,017,538, which is in turn a nationalphase of International Application PCT/FR93/01024, filed Oct. 18, 1993,which in turn claims foreign priority to the French Application 9212488,filed Oct. 19, 1992.

BACKGROUND OF THE INVENTION

The object of the present invention is novel preparations for abroad-spectrum antiplasmodial vaccine.

The object of the invention is also a vaccinating antigen of Plasmodiumfalciparum capable of inducing a resistance to the parasite whichreproduces that observed in the mechanism of protective immunity orpremunition.

The object of the invention is also preparations of monoclional orpoyclonal antibodies or chimeric fragments obtained from theseantibodies specific for these antigens and likely to form part of acomposition for passive immunotherapy.

Finally, the object of the invention is a kit permitting the in vitrodiagnosis of the infection of an individual by a broad spectrum ofplasmodial strains.

DESCRIPTION OF THE RELATED ART

The parasites responsible for malaria in man, including in particularPlasmodium falciparum or Plasmodium vivax to mention only the principalones, exhibit different morphologies in the human host and expressdifferent antigens as a function of their localization in the organismof the infected host. The morphological and antigenic differences ofthese parasites during their life cycle in man enable at least fourdistinct stages d development to be defined.

The very first stage of development of the parasite in man correspondsto the sporozoite form introduced into the blood of the host by bites ofinsect vectors of the parasite. The second stage corresponds to thepassage of the parasite into the liver and to the infection of thehepatic cells in which the parasites develop to form the hepaticschizonts which, when they are mature (for example in the case of P.falciparum on the 6th day after penetration of the sporozoite) releasehepatic merozoites by bursting. The third stage is characterized by theinfection of the blood erythrocytes by the asexual forms (merozoites) ofthe parasite; this erythrocytic stage of development corresponds to thepathogenic phase of the disease. The fourth stage corresponds to theformation of the forms with sexual potential (or gametocytes) which willbecome extracellular sexual forms or gametes in the mosquito.

It is known that very many studies have been undertaken to isolate fromstrains of parasites which infect a human host polypeptide fractions topermit the in vitro diagnosis of malaria by the detection of thecorresponding antibodies, on the one hand, and to attempt to vaccinateagainst malaria, on the other.

In 1976 the maintenance (so long-awaited) of P. falciparum in continuousculture in human RBC was accomplished (Trager and Jensen, Science 1976,193: 673; Haynes et al., 1976). This achievement facilitated access tothe parasite considerably and stimulated research which since then hasexperienced a rapid development. Efforts have been oriented mainlytowards the development of a vaccine which in future will be necessaryto control malaria whose incidence is becoming worse in as much asresistance of the parasite to drugs is spreading in different parts ofthe world.

In the search for a vaccine against the agent responsible for malaria,biologists are confronted with various problems not observed with otherinfectious agents such as viruses or bacteria. Of these specialdifficulties with the parasite we will mention principally:

The complexity Of the biological cycle of the plasmodium taking place intwo different hosts, the mosquito and man, undergoing sexualreproduction in the one and 2 different phases of asexual multiplicationin the other. Thus 2 stages take place in man differing in their site ofdevelopment (the liver and the blood circulation) and in their antigenicspecificities.

The antigenic diversity of the parasite Since 1983 the plasmodialantigens have been cloned and their nucleotide and protein sequenceshave been analyzed. This detailed study shows that more than 50% of theknown antigens exhibit a high degree of polymorphism from one strain toanother.

At the immunological level, the host-parasite relationship is verysubtle As has already been mentioned, for a given parasite it is verydifferent depending on the host in which it evolves. This leads to thedifficulty of interpretation of the results obtained in the experimentalmodels.

Furthermore, in the natural infection sterilizing immunity is never seenlike that observed for example in viroses. However, there is no doubtthat an acquired immunity exists but it is partial and labile.

Thus the complexity and the diversity of the parasite as well as theunusual nature of the immune response that it elicits are the majorreasons for the absence of an anti-malarial vaccine at present.

The research approach most often taken in the development of a vaccineagainst malaria due to P. falciparum hence consists of theidentification (on the basis of the information cited above) of apotential candidate, and then the evaluation of its value either invitro by testing the specific antibodies in the inhibition of the growthof the parasite or of certain of its properties (cytoadhesion, rosetteformation . . . ), or in vivo by the immunization of monkeys often withthe complete Freund adjuvant. The present situation may thus be summedup as the existence of a large number of potential candidatescharacterized by their biochemical properties, their nucleotide andprotein sequences, their degree of polymorphism, their localization onthe parasite etc. Nevertheless, the researchers dispose of limited meansfor assessing the value of their candidates: 1) in vitro testsimplicating mechanisms of action of antibodies whose validity in vivo ispoorly documented, 2) vaccinations of non-human primates, and hence theevaluation of the effect of a vaccine on an experimental infection isbased an parasitological and clinical parameters and particularly thetype of immunity which may be induced which are very different fromthose of the natural infection in man.

The strict specificity of the host-parasite relationship leads undernatural conditions to the opposite of what is ed in the animal models,to an equilibrium in which the parasite survives by inducing in itsnatural host a non-sterilizing immunity. The chronic nature of theparasitic infection suggests that the majority of the molecularcomponents of the parasite are selected so as to protect themicro-organism against the immune defences of the individual infected,and do so by means of escape which are very varied but specificallyadapted to the natural host. In the experimental host, the poorlyadapted parasite defends itself less well against the immune system andprotection against a single treated infection is easy to obtain, andvaccination is still easier to obtain.

Gordon-Thomson, Immunity in Malaria, Trans. Ray.Soc. Trop. Med. Hyg.XXVI (6) 483-514) dearly concluded that immunity against P. falciparumcan only be acquired in the regions where transmission is essentiallycontinuous year after year This “tolerance” to parasitism requires atthe individual level an uninterrupted infection for about 15 years,sometimes 20 years and up to 26 years in a study conducted in Panama Animmunity associated with a latent infection necessary for themaintenance of the protection results from this. Sergent (1935).Institut Pasteur d'Algerie Archives, 3:279 suggested the term“premunition” to define this “particular state or resistancecontemporaneous with the infection and ceasing with it”.

Thus the immunity (or premunition) against P. falciparum acquired by manin a holo- or hyperendemic zone is characterized by:

a very long delay prior to its installation (15 to 20 years ofinfection)

its incapacity to abolish the infection, it is a non-sterilizingimmunity.

its lability. In the absence of any reinfection (during more than oneyear), the premonition is lost and the subject again becomes susceptibleto the disease if subject to a new infection.

The indications in favour of humoral immunity in acquired protectionagainst malaria cone from the first attempts at passive transfer ofserum from an individual in the “chronic” phase who had reached a stateof premonition (i.e. showing circulating parasites in small numberswithout any clinical manifestation) to a subject in the acute phase. Thecondition of this latter is found to be improved subsequent to thispassive transfer (Sotiriades 1917, Attempts at serotherapy in malariaGreek Med. XIX: 27-28).

The role of antibodies in premunition is demonstrated by severalexperiments of passive transfer carried out at the beginning of the1960s. The transfer of IgG purified from hyperimmune African adult serumcures child victims of an acute infection by appreciably reducing theirparasitemia (Cohen et al., 1971, Trans, Roy. Soc. Trop. Med. Hyg. 65(2): 125-135; McGregor et al., 1964, the passive transfer of humanmaterial immunity, Am. J. Trop. Med. Hyg. 13: 237-239). The newborn areprotected up to the third month of their life as a result of maternalantibodies; this is proved by the beneficial effect of the IgG of theumbilical card transferred to children suffering from an acute attackdue to P. falciparum (Edozien et al., 1962).

The development of immunity and its efficacy in the protection of managainst P. falciparum nonetheless proves the existence of parasitemolecules which are the targets of an effective immune defence.

Recent experiments have made it possible to show that a) the Gimmunoglobulins (IgG) of immune African adults are protective by passivetransfer in man infected with malaria (Sabchareon et al., Amer. J. ofTrop. Med. and Hyg., vol. 45, No.3, Sept. 1991, 297-308), b) that,contrary to what is believed to be established, these antibodies areincapable of directly inhibiting the invasion of red cells by theparasites; on the other hand, they act by an antibody-dependent cellularinhibition mechanism (ADCI) in which the monocyte plays the role ofeffector cell (Bouharoun-Tayoun et al., J. Exp. Med, vol. 172, Dec.1990pp. 1633-1641; S. KHusmith et al., 1983, Inf. Imm. 41 (1): 219 and F.Lunel et al., 1989 Inf. Imm. 57: 2043),

c) This mechanism necessarily implicates cytophilic antibodies, i.e.those capable of binding to the monocyte through their Fc receptor; infact, there has been observed in the serum of protected subjects aprevalence of cytophilic isotypes IgG1 and IgG3 and in non-protectedsubjects a preponderance of non-cytophilic classes, IgG2 and/or IgM (H.BOUHAROUN-TAYOUN et al., 1992, Infection and Immunity, pp. 1473-1481).

SUMMARY OF THE INVENTION

One of the objectives of the present invention is the development ofpolypeptide for the vaccination of humans against malaria, polypeptideswhich are a target of the defence mechanisms prevailing in theindividuals having acquired an immunity by prolonged exposure to theparasite and their use in a vaccine by attempting to reproduce the samestate of resistance by the same mechanism as that observed in theestablishment of protective immunity.

The object of the invention is also the use of these same polypeptide inan in vitro diagnostic kit for the infection in man by a broad spectrumof plasmodial strains.

The invention relates more particularly to molecules or peptide orpolypeptide compositions characterized by the presence in theirstructure of one or more peptide sequences bearing one or more epitopescharacteristic of a protein recognized by antibodies of the cytophilicclass, i.e. capable of binding to the for receptors of the monocytesthrough their Fc region, and not recognized by non-cytophilic antibodiesand of promoting an antibody-dependent cytotoxicity mechanism (ADCI).

A protein of the invention is a merozoite surface protein of 48,000molecular weight (48 kD), exhibiting the properties given below.

The polypeptides of the invention were obtained by

the identification of a part of this protein of 48.000 daltons molecularweight (48 kD) Of the merozoite surface, this identification beingdescribed below,

the biochemical and immunological characterization of the 48 kD protein,

the screening of a genomic library of the plasmodium for its capacity toinhibit the coupling of a specific monoclonal antibody of the IgM typewhich has the special characteristic of blocking the ADCI-type reaction(“antibody-dependent cellular inhibition”) induced by the specific IgGof the plasma of the subjects protected by premunition

the characterization of the proteins synthesized by the clones selected,

the sequencing of the insert of the clone selected

the search for the functional effect of the antibodies corresponding tothis protein in the tests described.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the detection of the parasite protein of 48,000 (indicatedby the arrows) by immunoblot. The reactivity of the serum of miceimmunized with DG210 is studied in immunotransfer on the antigens of theblood stages of P. falciparum extracted into SDS (anti-R210 sera) orinto Triton-X114 detergent phase (A) and aqueous phase (B). Thereactivity of the human sera is studied on the SDS extracts by revealingthe isotopes IgG1 (1), IgG2 (2), IgG3 (3) and IgG4 (4). HIS: hyperimmuneserum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The value of the proteins and peptides of the invention and the strategyused to obtain them are made explicit in the description below.

Selection Strategy for the Proteins and Peptides

1—In Infection and Immunity (pp1473-1481, April 1992), the authors studythe isotypic distribution of individuals infected by the plasmodiumexhibiting various immunological states In this way they have shown thatthe unprotected subjects have an anti-plasmodial plasma antibodycomposition very much in favour of the non-cytophilic isotypes, namelyIgG2 and IgM. In certain cases, this equilibrium relates to theantibodies against all of the malarial polypeptides detectable byWestern blot (procedure described in Molecular Cloning 1989, Sambrook etal.) whereas in other cases it was possible to demonstrate IgG2 specificfor a given polypeptide, often a polypeptide of 48 kD appearing incertain isolates in the form of a dimer or a polypeptide of 80-100 kD.On the other hand, the polypeptide of 48 kD is always recognized by thecytophilic isotypes IgG1 and IgG3 in adults who have acquired resistanceto the disease, or a state of premunition.

2—It has often been observed in competition experiments that the totalpurified Ig of unprotected individuals block the ADCI reaction (seedescription below) induced by the IgG of resistant subjects This resultsuggests that the unprotected subjects have developed antibodiesdirected against the same epitopes as those which are recognized by theprotecting antibodies, but owing to the non-cytophilic character of theIgG2 or IgM derived from the unprotected subjects, these antibodies areincapable of promoting the destructive effect of the monocytes but, onthe other hand, are capable of entering into competition with antibodieseffective in ADCI When such a competitive effect was identified by usinghuman sera in which the antibodies against the 48 kD protein werepredominantly of the IgG2 isotype, that clearly demonstrates thesignificance of this 48 kD protein.

The ADCI test has already been described in the publication cited above(H. BOUHAROUN-TAYOUN et al, Khusmith et al., Lunel et al.). Briefly, itis a test of the inhibition of growth of the parasite by the IgG in thepresence of monocytes. The monocytes are isolated by adhesion to theplastic (in a 96-wells plate) from the fraction of mononucleated cellsof the peripheral blood of a normal donor. A synchronous culture of P.falciparum at 0.5% parasitemia in mature form is added to the monocytesin a monocytes/ red cells ratio of about 1/200. The hematocrit being 2%,the medium is supplemented by the serum or the IgG to be tested. Thereference cultures consist of parasites in the presence of normal IgG,parasites in the presence of manocytes and normal IgG, parasites in thepresence of the IgG to be tested.

Depending on the case, the culture will be stopped after 24, 48, 72 or96 hours. In the last two cases, 50 microliters of the culture mediumare added. The final parasitemia in each of the wells is estimated bycounting 10.000 red cells on stained smears. The results are presentedin the form of a specific growth inhibition index (SI) expressedas apercentage and calculated as follows, taking into consideration of thepossible inhibitory effect on the monocyte culture and/Cr antibodiealone: ${SI} = \frac{\begin{matrix}{1 - {\% \quad {parasitemia}\quad {culture}} + {{IgG}\quad {{test}/}}} \\{{\% \quad {parasitemia}\quad {monocytes}} + {{IgG}\quad {test}}}\end{matrix}}{{\% \quad {parasitemia}\quad {monocytes}} + {{{IgG}/\%}\quad {parasitemia}\quad {culture}} + {IgG}}$

This selection strategy for a potentially vaccinating protein of 48 kDaccording to the recognition criteria by cytophilic antibodies inprotected subjects and non-cytophilic antibodies in unprotected subjectsas well as by their capacity to induce antibodies capable of cooperatingwith monocytes in ADCI have led us to select this 48 kD protein orpeptides representing the epitopic regions of this protein aspotentially very useful candidates for inducing the protectiveimmunological effect against infections of P. falciparum in patients.

The invention also relates particularly to molecules or peptide orpolypeptide compositions characterized by the presence in theirstructure of one or more peptide sequences bearing one or more epitopescharacteristic of the protein and meeting the three criteria:

recognition by antibodies of the cytophilic class in protected subjectsand non-cytophilic in unprotected subjects,

their capacity to induce antibodies capable of cooperating withmonocytes in ADCI,

their slight or non-existent polymorphic character in as much as theprotective immunity can be exercised with respect to a large number ofstrains.

The molecules of the invention are all at the molecules bearingepitopes, recognized by antibodies recognizing epitopes borne by the 48kD protein of the merozoite surface.

A polypeptide composition according to the invention is characterized bythe presence of a sequence of 64 amino acids or a derivative sequencepossessing the same antigenic properties, and an example of which isgiven in the following formula I (SEQ ID NO:1):

his glu arg ala lys asn ala tyr gln lys ala asn gln ala val leu lys alalys glu ala ser ser tyr asp tyr ile leu gly trp glu phe gly gly gly valpro glu his lys lys glu glu asn met leu ser his leu tyr val ser ser lysasp lys glu asn ile ser lys glu asn glu

The invention relates primarily to synthetic monomeric peptidescomprising a unique peptide sequence of 64 amino acids correspondingrespectively to the formula indicated above and whose terminal aminoadds possess free amino and carboxylic extremities respectively, oroligomers containing in particular multiples of any one of theabove-mentioned peptide sequence.

It is obvious that the free reactive functions which some amino acidsforming part of the constitution of the molecules according to theinvention are likely to possess, in particular the free carboxyl groupsborne by the Glu residues and by the C-terminal amino acid, on the onehand, and/or the free groups bone by the N-terminal amino acid or byamino acids in the interior of the peptide chain, for example Lys, onthe other, may be modified provided that this modification does not leadto a modification of the antigenic, possibly immunogenic properties ofthe entire molecule. The molecules thus modified are naturally includedin the framework of the protection given to the invention by the Claims.These carboxylic functions are possibly acylated or esterified.

Other modifications are also included in the framework of the invention.In particular, amine or ester functions, or both at once, of theterminal amino acids may be themselves be engaged in linkages with otheramino acids For example, the N-terminal amino acid may be linked to asequence comprising one or more amino acids corresponding to a part ofthe C-terminal region of another peptide conforming to the definitionwhich has been given to it above, or vice versa.

It will be obvious that any peptide sequence derived from themodification of the peptide sequence of 64 amino acids by substitutionand/or by addition and/or deletion of one or more amino acids isincluded in the framework of the protection given to the invention bythe Claims, provided that this modification does not impair theantigenic or immunogenic properties of the pciypeptide, in particularwhen these immunogenic properties have been reinforced adequately, forexample by combination of this polypeptide with a suitable immunologicaladjuvant (for example, a muramylpeptide) or by coupling with a carriermolecule of higher molecular weight (for example a serum albumin or apolylysine ) or a toxin of the tetanic type or another antigen of P.falciparum.

The invention relates more generally to any molecule characterized bythe presence in its structure of One or more peptide sequencesexhibiting immunological cross-reactions with the peptide sequencecorresponding to the preceding formula with respect to the antibodiesinducible by these latter in vivo.

The invention also relates to any peptide whose structure is derivedfrom the preceding one and in particular to one of the three peptides offormula II, III, IV (SEQ ID NO:2-4).

his glu arg ala lys asn ala tyr gln lys ala asn II gln ala val leu lysglu ala ser ser tyr asp ala lys glu ala ser ser tyr asp tyr ile leu glyIII trp glu phe gly gly gly val pro glu his lys lys glu glu asn pro gluhis lys lys glu glu asn met leu ser his IV leu tyr val ser ser lys asplys glu asn ile ser lys glu asn glu

As in the case of the first peptide defined above, the differentpeptides which have just be named may be modified without being excludedfrom the framework of the invention provided that these structuralmodifications do not lead to major changes in their antigenicproperties.

The peptides according to the invention may be prepared by the standardprocedures used in the field of peptide synthesis. This synthesis may becarried out in homogeneous solution or on a solid phase.

For example, recourse may be had to the procedure of synthesis in ahomogeneous solution described by HOUBENWEYL in the monograph entitled“Methoden der Organischen Chemie” (Methods in Organic Chemistry) editedby E. Wunsch, vol. 15-I and II, THIEME, Stuttgart 1974.

This method of synthesis consists of successively condensing in therequired order the successive amino acids or of condensing peptidefragments previously formed already containing several amino acids inthe required order, it being understood that care will be taken toprotect temporarily all of the reactive functions borne by these aminoacids or peptide fragments, with the exception of those amino andcarboxyl functions which are necessarily implicated in the formation ofthe peptide bonds, in particular after activation of the said carboxylfunctions, according to the methods well-known in peptide synthesis, Asan alternative, it is possible to have recourse to coupling reactionsinvolving standard coupling reagents of the carbodiimide type, such asfor example 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide.

When the aminoacyl residue to be coupled possesses an additional acidicfunction (in particular in the case of glutamic acid), these functionsare protected, for example, by means of t-butyl ester groups.

In the case of stepwise synthesis, amino acid by amino acid, thesynthesis starts preferably by the condensation to the C-terminal aminoacid of the next amino acid in the desired sequence and so on, one at atime, the other amino acids selected in appropriate sequence, thesynthesis being completed with the attachment of the N-terminal aminoacid. According to another preferred procedure of the invention recourseis had to the procedure described by R. D. MERRIFIELD in the articleentitled “Solid phase peptide synthesis” (J. Am. Soc, 45: 2149-2154).

In order to synthesize a peptide chain according to the MERRIFIELDprocedure, the first amino acid, the C-terminal amino acid of the chain,is attached to a very porous resin polymer. This amino acid is bound tothe resin through the intermediary of its carboxyl group and its aminofunction is protected, for example, by the t-butoxycarbonyl group.

When the first, C-terminal amino add has thus been attached to theresin, the protecting group of the amino function is removed by washingthe resin with acid.

In the case in which the protecting group of the amine function is thet-butoxycarbonyl group, it may be removed by treatment of the resin withtrifluoroacetic acid.

The second amino acid is then coupled to the deprotected amine functionof the first C-terminal amino acid to furnish the second aminoacylresidue of the desired sequence, counting from the C-terminus.Preferably, the carboxyl function of this second amino add is activatedfor example by means of dicyclohexyl-carbodiimide and the amine functionis protected, for example by means of the t-butoxycarbonyl group.

The first part of the desired peptide chain is thus obtained, whichcontains two amino acids and the terminal amino function of which isprotected. As previously, the amine function is deprotected and it isthen possible to proceed to the attachment of the third aminoacylresidue under conditions analogous to those for the addition of thesecond, penultimate C-terminal amino acid.

In this way, the amino acids which will constitute the peptide chain areadded one after the other to the previously deprotected amine group ofthe portion of the peptide chain already formed which is attached to theresin.

When the desired peptide chain has been assembled in its entirety, theprotecting groups of the different side chains of the amino acidsconstituting the peptide chain are removed and the peptide is cleavedfrom the resin, for example with the aid of hydrogen fluoride.

The invention also relates to water-soluble oligomers of the monomericpeptides indicated above.

The oligomerization may cause an increase in the immunogenicity of themonomeric peptides acording to the invention. Without such numericalvalues being considered as limiting, it should nonetheless be mentionedthat these oligomers may contain, for example, from 2 to 10 monomericunits.

The monomeric units forming part of this oligomer are either constitutedby the polypeptide of sequence I or by the polypeptides of sequence I,III or IV.

In order to carry out the oligomerization, recourse may be had to anypolymerization procedure currently used in the field of peptides, thispolymerization being conducted until an oligomer or polymer is obtainedwhich contains the required number of monomeric motifs for theacquisition of the desired immunogenicity.

One method of oligomerization of polymerization of the monomer consistsin the reaction of the latter with a cross-linking agent such asglutaraldehyde.

It is also possible to have recourse to other methods of oligomerizationor coupling, for example to that making use of the successive couplingof monomeric units through the intermediary of their terminal carboxyland amino functions in the presence of homo- or hetero-bifunctionalcoupling agents.

For the production of molecules containing one or more motifs of 64amino adds such as defined above it is also possible to have recourse togenetic engineering procedures making use of micro-organisms transformedby a specific nucleic acid comprising corresponding suitable nucleotidesequences.

Consequently, the invention also relates to nucleic acids containing oneor more of these sequences each comprising 64 triplets of the typeindicated above.

The invention also relates to the conjugates obtained by covalentcoupling of the peptides according to the invention (or theabove-mentioned oligomers) to carrier molecules (natural or synthetic),physiologically acceptable and non-toxic, through the intermediary ofcomplementary reactive groups borne respectively by the carrier moleculeand the peptide. Examples of suitable groups are illustrated in whatfollows:

As examples of carrier molecules or macromolecular supports forming partof the composition of the conjugates according to the invention, mentionshould be made of naturally occurring proteins such as tetanus toxoid,ovalbumin, serum albumin, hemocyanins, Mention should be made, forexample, of polylysine or poly (D-L-alanine)-poly (L-lysine) as examplesof synthetic macromolecular supports.

The literature mentions other types of macromolecular supports which canbe used and which usually have a molecular weight higher than 20,000.

In order to synthesize the conjugates according to the invention,recourse may be had to known procedures such as that described by FRANTZand ROBERTSON in Infect. and Immunity, 33, 193-198 (1981) or thatdescribed in Applied and Environmental Microbiology, (October 1981),vol. 42, No. 4, 611-614 by P. E. KAUFFMAN by using the peptide and theappropriate carrier molecule.

In practice, the following compounds, cited in a non-limiting manner,are advantageously used as coupling agents: glutaraldehyde, ethylchloroformate, water-soluble carbodiimides: N-ethyl-N′(3-dimethylamino-propyl) carbodiimide HCl, diisocyanates,bis-diazobenzidine, di- and tri-chloro-s-triazines, cyanogen bromide aswill as the coupling agents mentioned in Scand. J. Immunol., (1978),vol. 8, p. 7-23 (AVRAMEAS, TERNYNCK, GUESDON).

It is possible to have recourse to any coupling procedure implicating onthe one hand, one or more reactive functions of the peptide and, on theother, one or more reactive functions of the molecular supports.Advantageously, these are carboxyl and amine functions which can giverise to a coupling reaction in the presence of a coupling agent of thetype used in the synthesis of proteins, for example,1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide N-hydroxybenzotriazole,etc. . . . It is also possible to have recourse to glutaraldehyde, inparticular when it is required to link together amino groups bone by thepeptide and the molecular support, respectively.

A group of preferred molecules according to the invention is constitutedof those possessing an alpha helical conformation, this latterreinforcing the antigenic and immunogenic properties of said molecules.Such molecules possessing an alpha helical conformation weredemonstrated by circular dichroism in trifluoroethanol or in aqueoussolution.

The molecules according to the invention possess antigenic propertiescharacteristic of the 48 kD antigen of the merozoite specific for theerythrocyte stage of the development of P. falciparum and exhibiting theparticular characteristics described above.

In fact, as will be more particularly described with the aid of examplesof molecules according to the invention in the detailed descriptionwhich follows, the molecules according to the invention reactspecifically with the anti-48 kD protein antibodies predominantly of theIgG2 or IgM isotype in the patients sensitive to the infection, andpredominantly of the IgG1 or IgG3 isotype in protected subjects.

These molecules according to the invention are capable of triggering invivo the synthesis of specific immunoglobulins, and are capable ofinducing in vivo the neutralization of the merozoite present in theblood, its process in the monocytes and the inactivation of theintraerythrocytic development of P. falciparum subsequent to aninteraction between the monocytes and the extra-erythrocytic freeparasites or merozoites through the intermediary of a cytophilicantibody by binding of the Fc fragment of the immunoglobulin to thegamma receptor of the monocyte.

FIG. 1 shows the detection of the parasite protein of 48,000 (indicatedby the arrows) by immunoblot. The reactivity of the serum of miceimmunized with DG210 is studied in immune transfer on the antigens ofthe blood stages of P. falciparum extracted into SDS (anti-R210 ser) orinto Triton-X114 detergent phase (A) and aqueous phase (B). Thereactivity of the human sera is studied on the SDS extracts by revealingthe isotypes IgG1 (1), IgG2 (2), IgG3 (3) and IgG4 (4). HIS: hyperimmuneserum.

EXAMPLE

In the following example as in all of the experiments described in thepresent description the immunoglobulins of human plasma are obtained bythe method described by A SABCHAREON et al., J. Trop. Med. Hyg. 1991, 45(3): 297). The ADCI test is described above.

In the following example the specific inhibition indices (S.I.) obtainedboth with sera of mice immunized with the peptide III and with immunehuman antibodies purified with the aid of an affinity column bearing thepeptide III (procedure described in OKAZI et al.) are compared. Both thesera and the antibodies are capable of recognizing the 48 kD proteinboth in indirect immunofluorescence and in Western blot tests, and do sounder the same conditions as previously (IgG2 of sensitive patients andIgG1 or IgG3 of protected patients). Finally, the immunopurifiedantibodies like the antibodies induced by injection of peptide II intothe mouse tested in ADCI tests confirm that they are capable of inducingthe inactivation of the parasite by the intermediary of the monocytes.

The following Table I summarizes the results in support of theseobservations

TABLE 1 Specific inhibition Antibodies index (%) Controls Pshi 60 (+)shi1 77 shi2 66 Controls spi 0 (−) anti-βgal −18 anti-DG210 45 Testanti-DG328 −13 anti-DG414 4 anti-210B1 72 B2 80 competitions Pshi +Acm245 20 Pshi + spi 23

in which Pshi represents a pool of hyperimmune serum, shi1 and 2 of thehyperimmune sera of two different donors, spi and anti-betagal, controlsderived from serum after a first invasion and an anti-betagal control;anti-DG210 are purified antibodies against peptide I, anti-210B (1) arepurified human antibodies against peptide III anti-210B (2) are theantibodies induced in the mouse and the anti-R328 and R414 are purifiedantibodies against peptides derived from other clones.

The specific inhibition index is that measured by the ADCI procedure.

The molecules acording to the invention are hence capable of inducingthe synthesis of antibodies of a class capable of cooperating withmonocytes.

The proteins and peptides of the invention are not limited to thoseparticularly described above.

The invention relates to all of the natural peptides or poly-peptidesobtained by genetic recombination or synthesis which exhibit the sameproperties of being capable of inducing immune defence mechanismsdeveloped and characteristic of the subjects protected by malaria.

As a result of this feature, the invention relates in particular toepitope of the 48 kD protein different from the polypeptides II, III andIV above In fact, we have been able to show that the immunoglobulins ofsome individuals read with an epitope of the 48 kD protein in Westernblot whereas these same immunoglobulins do not recognize the antigenexpressed by the done DG210.

The invention also relates to the poyclonal or monoclonal antibodiesexhibiting the characteristic of recognizing the molecules of theinvention and of cooperating with the monocytes, and capable of beingused in pharmaceutical compositions to protect infected subjects bypassive immunotherapy and presenting or being able to present thesymptoms of the disease.

The monoclonal antibodies may be produced by the hybridoma technique inaccordance with the standard procedures comprising:

the fusion of a myeloma cell with spleen cells of an animal previouslyimmunized with one of the antigens according to the invention,

the culture of the hybridomas formed by the fusion of the aforementionedcells and,

the selection of those hybridomas capable of foaming monoclonalantibodies recognizing the antigen used for the immunization of theanimals.

The animals selected for the immunization may be for example mice.

Of these monoclonal antibodies the cytophilic monoclonal antibodies willbe selected advantageously, i.e. those whose Fc fragment is capable ofbinding to the Fc receptor of the human monocytes.

Another procedure for the production of antibodies may enable humanmonoclonal antibodies to be formed in vitro. To do this, B lymphocytesimmortalised with, for example, the Epstein Barr virus are used. Theselymphocytes may be taken from a person having been infected by P.falciparum. In this case, they make possible the production ofmonoclonal antibodies against several antigens without having recourseto in vitro stimulation by novel antigens.

Another possibility consists in fusing B lymphocytes immortalised asdescribed above with human B lymphocytes stimulated in vitro beforehandwith an antigen according to the invention against which it is desiredto form monoclonal antibodies under culture conditions permitting thestimulation of the lymphocytes.

Reference will advantageously be made to the technique described byDesgranges C. et al. (1987, J. of Virological Methods, vol. 16,p281-292) for the preparation of the human monoclonal antibodies of theinvention.

It is also contemplated within the framework of the invention to producehuman monoclonal antibodies by genetic recombination by carrying out anin vitro transfection of the gene coding for the variable part of theantibody into vectors infecting bacteria under conditions permitting theexpression of a human immunoglobulin.

Finally, the present invention relates to any type of monoclonalantibody, chimeric or hybrid, or even any fragment of poyclonal ormonoclonal antibody of the Fab or Fab′2 type, and exhibiting the sameaffinity characteristics for the epitopes of the 48 kD protein or thepeptides I, II and III, IV below.

Preferred monoclonal antibodies according to the invention are humanantibodies of class IgG1 or IgG3, or antibodies obtained in animals andhaving cytophilic properties in man, directed against one or more of theantigens whose sequence was described above.

The invention also relates to a procedure for monitoring the vaccinationof the patient against infection with P. falciparum, starting from abiological sample such as blood, characterized in that it comprises:

the placing of the biological sample likely to contain protectiveantibodies against P. falciparum in contact with at least one antigenaccording to the invention,

the detection of the antigen-antibody reaction.

For carrying out this in vitro detection method the antigens accordingto the invention are advantageously labelled with the aid of aradioactive marker, an enzymatic or fluorescent label or even a physicaltype of marker.

The invention also relates to kits for the in vitro detection of thepresence of antibodies directed against the antigens of the invention,characterized in that they contain:

an antigenic composition including at least one antigen according to theinvention,

reagents necessary for carrying out the immunological reaction betweenthe above-mentioned antigens and the antibodies possibly present in thebiological sample,

reagents making possible the detection of the antigen-antibody complexproduced by the immunological reaction.

These reagents are for example labelled or capable of being recognizedby a labelled reagent.

II—Isolation of the clone DG 210:

a) Construction of the Library

A DNA genomic bank was constructed in the expression bacteriophage λgt11by using the genomic DNA of the clone Tak 9-96 of P. falciparum (ref.done Tak 9-96: Science 212, 137; 1981) in accordance with the protocoldescribed in the detail in the EP patent application of Feb. 9, 1987published under the number 0343186.

Briefly, the DNA was excised by DNAase I in the presence of Mn2+ions,methylated by EcoRI methylase to protect the natural EcoRI sites thenrepaired by the DNA polymerase of the T4 bacteriophage and the DNAligase of EcoRI. EcoRI “linkers” (synthetic oligomers) were ligated tothe DNA fragments of P. falciparum and the artifical sites thus addedwere released by cutting with the enzyme EcoRI The fragments werepurified on a sucrose gradient and ligated to the DNA of the vectorλgt11 suitably prepared (i.e. cut with EcoRI and dephosphorylated—soldby Promega Biotec). The DNA was encapsidated in vitro in viralparticles. The bacteriophages derived from this procedure constitute agenomic DNA library.

b) Immunological Screening of the Bank

The technical details of screening are given in the text of the patentapplication 034186. Of a series of monoclonal antibodies (Mabs) usedpreviously, Mab 245 (Soulier et al., Revue Francaise de Transfusion etImmunohématologie, Tome XXV, No. 4, 1982, page 373) of class IgM, aclass of antibodies incapable of cooperating with the monocytes is theonly one which has proved capable of entering into competition with thepolyclonal antibodies of an immune subject active in the ADCI test, i.e.of appreciably reducing the inhibitory effect of these antibodies inADCI, suggesting that the target epitope of these antibodies capable ofcooperating with the monocytes and of this Mab 245 is identical. It isthis antibody which was used for the isolation of the gene by screeningof a band of genomic DNA cloned in the expression vector λgt11.

A direct screening by antigen/antibody reaction with the proteinssynthesized by the clones of the library proved to be unsuccessful.Since this Mab is capable of entering into competition with otherantibodies for a epitope bone by the parasite protein, another method ofscreening was then used.

The recombinant antigens were screened by a competition test usingindirect immunofluorescence The monoclonal antibody Mab 243 in thepresence of the merozoite was incubated with each of the recombinantantigens (supernatant of different clones of the genomic band) and theinhibition of the binding of the antibody to the parasite was measuredby indirect immunofluorescence (technique described in H.BOUHAROUN-TAYOUN et al., 1990, J. Exp. Med. 172: 1633-1641).

Six antigens proved positive, i.e. inhibitory, and were studied indetail. These six protein antigens thus selected were bound to resins inorder to effect affinity purifications of the poyclonal antibodiesderived from immune human sera according to the technique described byOKAZI et al. These immunoglobulins thus purified were studied. Among thelatter, the one obtained by binding to the protein synthesized by thedone DG 210 recognizes in Western blot the 48 kD dimer which appears tobe identical with that recognized by the cytophilic classes of IgG foundin adult subjects in a state of clinical resistance to the disease andby the non-cytophilic classes of the sensitive individuals. On the otherhand, it is different from the antigen MSA2, a surface antigen of themerozoite which on the same gene appears as a pciypeptide of highermolecular weight (Figures). The results of Table I show that theantibodies isolated by immunoaffinity to the protein secreted by theclone DG 210 are capable of promoting in vitro the inhibitory effect onthe growth of the trophozoite induced by the manocytes by the ADCIprocedure.

The clone DG 210 was deposited with the CNCM on Oct. 19, 1992 under thenumber No. I-1270.

Characterization of the Protein Synthesized by the Clone DG 210

The human antibodies immunoabsorbed on this protein like those producedin the mouse by immunization with the done DG 210 show in indirectfluorescence an image in clusters designating the circumference of themerozoites within the mature intra-erythrocytic schizonts. Thisindication that the molecule is localized at the membrane of themerozoites was confirmed on the one hand by extraction with a non-ionicdetergent, Triton X114, from purified merozoites and detection of theprotein in the soluble “detergent” phase on the other, by the action ofphospholipase C of Bacillus aureus, this enzyme releasing the proteinfrom a preparation of purified merozoites which thus indicates that thelatter is anchored by a phosphatidyl-inositol group; finally, byrevelation of the localization of the antibodies in electron microscopywith the aid of a second antibody labelled with colloidal gold: theseantibodies are directed mainly against an antigen situated at thesurface of the merozoites of P. falciparum.

These results confirm that the antigen capable of stimulating theantibody-dependent cytotoxicity mechanism (ADCI) is situated at thesurface of the extracellular form of the parasite, the merozoite, Inaddition, the antibodies obtained by immunoaffinity on the recombinantproduct of the clone DG 210 have a very high inhibitory potency towardsthe growth of P. falciparum in the ADCI test whereas these sameantibodies have no effect on the infection of the red cell by themerozoite The antibody controls prepared in the same manner with othercontrol recombinant proteins including MSA2 and RESA had no inhibitoryeffect either directly or in the ADCI assays (Figure). The results arefound in three separate experiments involving three different isolatesof antibodies. Two of these results are shown in FIG. 1.

These results are confirmed by complementary observations. The isotypicdistribution of the antibodies directed against the recombinant proteinderived from the clone DG 210 exhibits the following characteristics.IgG2 isotypes are found much more abundantly in the unprotected patientswhereas the protein is recognized preferentially by cytophilic IgG1 andIgG3 in the blood of protected subjects. Thus, the epitopes contained inthe recombinant protein of the done DG 210 have all the desiredcharacteristics for a protein with a vaccinating effect, namely thatthey might induce in vivo non-cytophilic antibodies in unprotectedsubjects which on the other hand, are cytophilic in protected subjectsand thus are capable of inducing the ADCI reaction in vivo.

Finally, the study of the lymphoproliferative response of 70 subjectsexposed to malaria (in Senegal and Madagascar) reveals that the peptidesII, III and IV define epitopes recognized by the T lymphocytes.

A strong prevalence of lymphoproliferative responses (>50% of thepopulation study) was observed in these subjects exposed to the disease.

Sequencing and Characterization of the Genome of the Clone DG 210

The genome of the done DG 210 has a length of 1300 base pairs. It waspossible to determine its size by using the method described byMcCutchan (McCutchan et al. (1984), Science 225: 625-627). Briefly, thegenome is digested by the Mung bean endonuclease, the restrictionfragments are then hybridized with the DG 210 probe labelled withphosphorus 32, and revealed in autoradiography according to procedureswell-known to specialists skilled in the art.

The “North Blot” study of these same fragments and revealed by the sameradioactive probe confirms that the gene is expressed during theerythrocytic phase of the cycle of the parasite.

The analysis of the sequence of 192 base pairs of the insert was carriedout by the method of Sanger et al. (PNAS, 74: 5463, 1977), called“dideoxy-termination” method.

The invention also relates to the recombinant nucleic acids containingat least one of the polypeptide sequences I, II, III or IV or acombination of these as well as to the micro-organisms, in particularthe E. coli bacteria, transformed by these recombinant nucleic acids andcapable of expressing said polypeptide.

The invention relates to these nucleic acid sequences or equivalentsequences which can be synthesized and which code for the same aminoacids.

It will be immediately apparent to the person skilled in the art that inthese sequences some of the nucleotides may be replaced by others onaccount of the degeneracy of the genetic code without the encodedpeptides being modified. All of these nucleotide sequences, as well asthose which code for polypeptide which differ from the foregoing by oneor more amino acids without their intrinsic immunogenic activity beingsimilarly modified form part of the invention. Obviously the same holdsfor the nucleotide sequences which may be reconstituted and which arecapable of coding for oligomers such as defined above The monomericmotifs are directly linked end-to-end or through the intermediary ofpeptide; sequences without effect on the immunogenic properties of theoligomers thus formed.

Finally, the invention relates to the vectors modified by thesesequences, these vectors being naturally provided with regulatory andtermination elements preceding and following the above-mentioned nucleicacid sequences which will permit the expression of these latter incompetent cellular organisms.

Among the nucleotide sequences which code for the characteristicpeptides which have been defined above, mention should be made of thosewhich are characterized by the triplet sequences which follow (SEQ IDNO:5-8), these sequences corresponding in particular for the first topeptide I and for the three others to peptides II, III and IV whoseformulae were indicated previously

CAT GAA AGG GCA AAA AAT GCT TAT CAA AAA (1) GCA AAC CAA GCT GTT TTA AAAGCA AAA GAA GCT TCT AGT TAT GAT TAT ATT TTA GGT TGG GAA TTT GGA GGA GGCGTT CCA GAA CAC AAA AAA GAA GAA AAT ATG TTA TCA CAT TTA TAT GTT TCT TCAAAG GAT AAG GAA AAT ATA TCT AAG GAA AAT GAG CAT GAA AGG GCA AAA AAT GCTTAT CAA AAA (2) GCA AAC CAA GCT GTT TTA AAA GCA AAA GAA GCT TCT AGT TATGAT GCA AAA GAA GCT TCT AGT TAT GAT TAT ATT (3) TTA GGT TGG GAA TTT GGAGGA GGC GTT CCA GAA CAC AAA AAA GAA GAA AAT CCA GAA CAC AAA AAA GAA GAAAAT ATG TTA (4) TCA CAT TTA TAT GTT TCT TCA AAG GAT AAG GAA AAT ATA TCTAAG GAA AAT GAG

Bacteria harbouring the above-mentioned clones DG 210 were depositedwith the Collection Nationale des Cultures de Microorganismes (CNCM) atthe Pasteur Institute in Paris on Oct. 19, 1992 under the number I-1270.

The object of the invention is also DNA or RNA primers utilizable forexample in the framework of the synthesis of nucleotide sequences,possibly flowed by polypeptide synthesis, according to the invention bythe PCR (Polymerase Chain Reaction) procedure such as described in theAmerican patents Nos. 4683212 and 4683195 and the European patentapplication No. 200362. A description of the procedure used here isfound in the PCT patent application No. FR 91/00639, pages 28 to 30.

The peptides of the invention can also be prepared by the standardprocedures used in the field of peptide synthesis. This synthesis may becarried out in homogeneous solution or on a solid phase such asdescribed above by the procedures described in HOUBENWEYL or MERRIFIELD.

III—Study of the Polymorphism of the Gene and Epitopes Defined by theClone DG 210

A major impediment to the production of an effective vaccine is, inaddition to the complexity of the cycle of the parasite, its antigenicdiversity and the high degree of polymorphism from one strain toanother.

The conservation of the gene and defined epitopes in the clone DG 210has been studied by several procedures in a series of isolates ofplasmodiae.

By using the following nucleotides as primers: (SEQ ID NO:9-10)

GAA AGG GCA AS A AAT GCT TAT (5)

or TAA AAG GAA TCT ATA TAA AAG (6)

the DNA fragments of two cultures o cultured strains of African P.falciparum, of 4 Thai isolates and 29 African isolates could beamplified by the PCR procedure.

The corresponding gene was present everywhere, with no apparent sizepolymorphism whereas a similar experiment using the same PCR procedurewith primers of the MSA1 and MSA2 regions could not demonstrate thisresult.

Similarly, the screening of the proteins and peptides by Western blotprepared from 6 Thai or African isolates with antibodies purified usingan affinity column with the peptide 210 as ligand have enabled the 48 kDdimer to be revealed in all the variants, with no change of molecularweight from one isolate to another.

Finally, 10 isolates from the Congo were studied by means of indirectimmunofluorescence by the same procedure as above and were all positive,and all the parasites of each of the isolates were labelled with theantibodies purified by affinity.

Everything thus seems to point to the absence of antigenic polymorphismat least in the region of the molecule bearing the epitope B. just likethe conservation of the size of this protein from one isolate toanother.

These results confirm those obtained in ADCI, and more particularly inthe competition tests in which the non-cytophilic antibodies obtainedafter an initial attack by the parasite are excellent competitors of thecytophilic antibodies of the protected adults.

In as much as the non-cytophilic antibodies obtained after the initialattack correspond to a single isolate, and the protected adults areprotected against the infection of a large number of polymorphicisolates (which, furthermore, were isolated in the competitionexperiments), it is right to conclude that the epitopes concerned in thecompetition experiments are representative of non-polymorphic, conservedregions.

The polypeptides and proteins of the invention are hence characterizedby a broad activity spectrum as vaccinating composition.

10 64 amino acids amino acid single linear peptide 1 His Glu Arg Ala LysAsn Ala Tyr Gln Lys Ala Asn Gln Ala Val Leu 1 5 10 15 Lys Ala Lys GluAla Ser Ser Tyr Asp Tyr Ile Leu Gly Trp Glu Phe 20 25 30 Gly Gly Gly ValPro Glu His Lys Lys Glu Glu Asn Met Leu Ser His 35 40 45 Leu Tyr Val SerSer Lys Asp Lys Glu Asn Ile Ser Lys Glu Asn Glu 50 55 60 23 amino acidsamino acid single linear peptide 2 His Glu Arg Ala Lys Asn Ala Tyr GlnLys Ala Asn Gln Ala Val Leu 1 5 10 15 Lys Glu Ala Ser Ser Tyr Asp 20 27amino acids amino acid single linear peptide 3 Ala Lys Glu Ala Ser SerTyr Asp Tyr Ile Leu Gly Trp Glu Phe Gly 1 5 10 15 Gly Gly Val Pro GluHis Lys Lys Glu Glu Asn 20 25 28 amino acids amino acid single linearpeptide 4 Pro Glu His Lys Lys Glu Glu Asn Met Leu Ser His Leu Tyr ValSer 1 5 10 15 Ser Lys Asp Lys Glu Asn Ile Ser Lys Glu Asn Glu 20 25 192base pairs nucleic acid double linear DNA (genomic) 5 CATGAAAGGGCAAAAAATGC TTATCAAAAA GCAAACCAAG CTGTTTTAAA AGCAAAAGAA 60 GCTTCTAGTTATGATTATAT TTTAGGTTGG GAATTTGGAG GAGGCGTTCC AGAACACAAA 120 AAAGAAGAAAATATGTTATC ACATTTATAT GTTTCTTCAA AGGATAAGGA AAATATATCT 180 AAGGAAAATG AG192 75 base pairs nucleic acid double linear DNA (genomic) 6 CATGAAAAGGCAAAAAATGC TTATCAAAAA GCAAACCAAG CTGTTTTAAA AGCAAAAGAA 60 GCTTCTAGTTATGAT 75 81 base pairs nucleic acid double linear DNA (genomic) 7GCAAAAGAAG CTTCTAGTTA TGATTATATT TTAGGTTGGG AATTTGGAGG AGGCGTTCCA 60GAACACAAAA AAGAAGAAAA T 81 84 base pairs nucleic acid double linear DNA(genomic) 8 CCAGAACACA AAAAAGAAGA AAATATGTTA TCACATTTAT ATGTTTCTTCAAAGGATAAG 60 GAAAATATAT CTAAGGAAAA TGAG 84 21 base pairs nucleic acidsingle linear other nucleic acid /desc = “SYNTHETIC DNA PRIMER” 9GAAAGGGCAA AAAATGCTTA T 21 21 base pairs nucleic acid single linearother nucleic acid /desc = “SYNTHETIC DNA PRIMER” 10 TAAAAGGAATCTATATAAAA G 21

What is claimed is:
 1. An isolated DNA coding for a protein or peptidewhich is recognized by cytophilic antibodies of protected subjects (bypremonition) against infection by the Plasmodia and recognized bynon-cytophilic antibodies of subjects sensitive to the infection by thePlasmodia, wherein said cytophilic antibodies recognizing said proteinor peptide inhibit the growth of the Plasmodia in an antibody-dependentcellular inhibition test (ADCI), and wherein said protein or peptidecomprises SEQ ID NO:1.
 2. An isolated DNA coding for a protein having amolecular weight of 48,000 daltons +/−10% and which is a surface proteinof the merozoite of Plasmodium falciparum which is recognized bycytophilic antibodies of protected subjects (by premonition) againstinfection by the Plasmodia and recognized by non-cytophilic antibodiesof subjects sensitive to the infection by the Plasmodia, wherein saidcytophilic antibodies recognizing said protein or peptide inhibit thegrowth of the Plasmodia in an antibody-dependent cellular inhibitiontest (ADCI), and wherein the protein comprises a sequence selected fromthe group consisting of SEQ ID NO:2 SEQ ID NO:3, and SEQ ID NO:4.
 3. Anisolated nucleotide sequence comprising SEQ ID NO:
 5. 4. An isolatednucleotide sequence which induces the synthesis of a peptide exhibitingan antigen cross-reaction with the protein of 48,000 daltons of themerozoites of Plasmodium falciparum, and which is selected from thegroup consisting of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 5. Arecombinant DNA comprising the DNA of claim
 3. 6. A recombinant DNAcomprising at least one DNA of claim
 4. 7. A recombinant cloning vector,comprising the nucleotide sequence of claim
 3. 8. A recombinant cloningvector, comprising the nucleotide sequence of claim
 4. 9. Therecombinant cloning vector of claim 7, wherein the nucleotide sequenceis under the control of a promoter and regulatory elements homologous orheterologous vis-a-vis a host cell, for expression in the host cell. 10.The recombinant cloning vector of claim 8, wherein the nucleotidesequence is under the control of a promoter and regulatory elementshomologous or heterologous vis-a-vis a host cell, for expression in thehost cell.
 11. A recombinant expression vector, comprising thenucleotide sequence of claim
 3. 12. A recombinant expression vector,comprising the nucleotide sequence of claim
 4. 13. The recombinantexpression vector of claim 11, wherein the nucleotide sequence is underthe control of a promoter and regulatory elements homologous orheterologous vis-a-vis a host cell, for expression in the host cell. 14.The recombinant expression vector of claim 12, wherein the nucleotidesequence is under the control of a promoter and regulatory elementshomologous or heterologous vis-a-vis a host cell, for expression in thehost cell.
 15. A recombinant host cell, which is transformed by thevector of claim 9 or claim
 13. 16. The host cell of claim 15, which isselected from the group consisting of a bacterium, a yeast, an insectcell, and a mammalian cell.
 17. The host cell of claim 16, which is anE. coli cell or a CHO cell.
 18. A recombinant host cell, which istransformed by the vector of claim 10 or claim
 14. 19. The host cell ofclaim 18, which is selected from the group consisting of a bacterium, ayeast, an insect cell, and a mammalian cell.
 20. The host cell of claim19, which is an E. coli cell or a CHO cell.
 21. A recombinant expressionvector, comprising the nucleotide sequence of claim 1.